Proteins in live cells can be studied, after being tagged by an optically observable label, by optical methods. Often, the optically observable tag used is a fluorescent protein. Presence of a fluorescent protein tag then allows observations of presence and spatial distribution of the studied protein in live cells, using a fluorescence microscope. However, presence and spatial distribution of fluorescence alone generally do not allow monitoring functional activity of the studied protein, such as whether a receptor protein is activated, an ion channel open or closed, a transporter protein transporting, etc.
Our ability to observe functional activity of proteins in living cells is very limited. Available optical methods generally rely on one or more of three basic principles: 1) production of an optically detectable species through activation of transcription; 2) fluorescence lifetime imaging (FLIM); and 3) fluorescence resonance energy transfer (FRET). Although useful, all of these methods have their limitations. Transcription activation takes minutes to hours, which is too long to observe many systems. FLIM requires expensive equipment, is not very sensitive, and FLIM data cannot be interpreted in terms of protein structure. FRET requires two optically active molecules (e.g. fluorescent proteins), which often negatively affects function of the system studied. Furthermore, the observed transfer of fluorescence energy (FRET signal) is only a fraction of the total fluorescence, and so is often hard to detect on the background fluorescence from the two present fluorescent moieties.